One third of the US population suffers from chronic pain, but the existing treatment options are inadequate. The overuse of opioid painkillers has contributed to the current opioid epidemic crisis. In this Blueprint Neurotherapeutics Network application, we propose to bring a novel class of potent non-opioid painkillers to the clinic. Using high-resolution structures of human glycine receptors (hGlyRs), we discovered a unique drug-binding site in the transmembrane domain that is responsible for mediating the non-psychoactive analgesic effects of marijuana. Using an innovative ensemble-based virtual screening approach to target this unique site, followed by in vitro electrophysiology measurements of functional modulation and in vivo measurements of pain treatment in an inflammatory pain model in mice, we discovered a new molecular scaffold that is GlyR subtype specific. The top hits of this scaffold are 10-100 times more potent than morphine in suppressing inflammatory pain. Moreover, the top hits can reduce the morphine requirement by a factor of 50 to achieve the same level of analgesia. In the preparatory (UG3) phase of this project, we will establish a lead development team with BPN consultants and solidify the scaffold's clinical value by evaluating the efficacy of the top hits in treating neuropathic pain. We will then move to the drug discovery (UH3) phase to complete the hit-to-lead optimization via the following three specific aims: (1) Maximize the potency and efficacy of new hits through (a) in silico SAR exploration to improve ADMET profiles and optimize binding affinity, (b) synthesizing the designed analogs by BPN contractors, and (c) evaluating the synthesized analogs in vitro using electrophysiology as the primary assay and in vivo using rodent inflammatory and neuropathic pain models as secondary assays. (2) Perform ADMET assays for the top analogs, focusing on bioavailability profiling, brain-to-plasma distribution, microsomal stability, plasma protein binding, cytochrome P450 (CYP) induction and inhibition, Ames activity, and flow cytometry of toxic markers in the liver, spleen, blood, lung, kidney, and brain. (3) Establish specificity through in vivo pain model measurements in ?3- GlyR knockout mice using analogs with favorable ADMET profiles to confirm the intended target. Counter-screening of off-target effects on opioid receptors, cannabinoid receptors, and other ion channels will be measured by the psychoactive drug screening program (PDSP) at NIMH. We aim to identify a single lead compound for the FDA investigational new drug approval by the end of the UH3 phase.